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United States Department of Agriculture

Agricultural Research Service

2011 Annual Report

1a.Objectives (from AD-416)
The goals of this research are to evaluate the host pathogen relationship using genomics and immunologically-based approaches, and use this information to develop methods that complement and/or reduce drug intervention as a means to control gastrointestinal (GI) nematodes of cattle. In this regard, we will identify structural variations in the bovine genome that influence host resistance to GI nematodes, pinpoint functional and/or structural variations in parasite genes associated with the drug resistant phenotype, identify and enhance immune factors that regulate the host-pathogen interface as an alternative to anthelmintic treatment, and finally use this collective knowledge to develop integrated parasite control programs that incorporate marker-assisted selection schemes that reduce economic loss caused by the parasites. These goals will be attained through a systems-based approach that will employ molecular, immunological, and genomic techniques to studies at the gene, cell and whole animal levels.

1b.Approach (from AD-416)
Studies will focus on using genomic approaches to develop novel means to control parasitic diseases in cattle, and on-farm application of these techniques. These studies will investigate both the parasite genome and the host genome. Studies focusing on the parasite will evaluate genetic factors associated with drug-resistant and drug-susceptible phenotypes in parasitic nematodes. Investigations of the host genome will focus on the identification of host genetic loci that affect resistance to parasite infection and will develop and implement genome-wide selection or marker-assisted selection programs based on high density SNP information. Additionally the project will use SNP haplotypes to refine and characterize QTL for parasite resistance that were identified in earlier studies. At the same time studies will continue to characterize the host immune response to parasitic infection using microarrays and real time PCR to discern gene expression patterns in cattle demonstrating different levels of resistance and/or immunity to the parasites. Finally, the information gleaned from these studies will be applied on-farm through co-operative research efforts with producers to establish practical management programs to control and diminish the effects of parasites on production effeiciency.

3.Progress Report
A beef cattle survey was performed to identify the incidence of gastrointestinal (GI) nematode infections in U.S. cattle operations and to assess the effectiveness of commercial drugs to control these parasites. Results unequivocally demonstrated that overuse of drugs has.
1)increased treatment failures;.
2)changed the distribution of parasite species; and.
3)caused resistance to develop in parasite species previously responsive to treatment. Results also showed that certain nematode species believed to be innocuous can cause harm to animal health. This study was the first comprehensive look at nematode parasitism and drug resistance in cattle in any developed country in the world. The results will advance guidelines to help manage GI nematodes in the American cattle industry. A gene present in larval GI nematodes that affects host energy metabolism was evaluated. Sequence similarity to other genes suggests that this class of enzymes plays a role in controlling extracellular levels of ATP and ADP which are important in cell signaling and initiating the host immune response. This gene was found in Ostertagia, Haemonchus, and Teladorsagia species that infect cattle and sheep. The production of a functionally active gene permitted us to characterize the activity of the enzyme. We found it exclusively expressed in fourth-stage larvae, which do massive damage to the host stomach (abomasum) in the absence of any inflammation. We believe that the lack of inflammation is the result of the parasite’s ability to degrade ATP and ADP, which are important in the inflammatory response of the infected animal. Work will continue toward characterizing the host-pathogen interface with respect to this class of enzymes in parasites. Work has continued on sequencing the Ostertagia genome and Cooperia genomes. Both of these parasitic nematodes are important pathogens of cattle. Currently, sequences of the genes expressed during development have been produced for each of these parasite genera and comparative analyses are underway, along with a draft of the first manuscript. To date, analyses show that even though these genomes are large, only a few genes are conserved and exclusive to these parasitic nematodes when compared to free-living nematodes. As such, the free-living nematode, C. elegans is not a good model for understanding nematode parasitism. Studies were conducted to better understand molecular mechanisms associated with parasite resistance in cattle. We found that the receptors for vitamin D activation were upregulated only during a repeat infection and may play a role in acquired resistance to parasites. Nitric oxide (NO) synthase, an enzyme that is important in the production of NO and cell signaling, was also strongly induced only during re-infection, and affected several metabolic pathways. The rapid development of acquired resistance to some GI nematodes may help explain reduced pathogenicity in cattle. We also continued to study the dynamics of the microbes in the stomach (abomasum) of cattle in response to Ostertagia infections. Our findings suggest that interactions between parasites and intestinal microbiota help define host pathophysiology.

1. A national survey of cattle GI nematodes and the effectiveness of anthelmintic treatment was completed. This work was performed in collaboration with APHIS and two University collaborators. Prior to this study, drug resistance had been observed predominantly in nematode parasites of sheep and goats, but only anecdotal information pointed to resistance in cattle species. In our study, cattle operations randomly selected throughout the U.S. participated in an effort to assess the mode, regiment, and effectiveness of drug treatment, and its relationship to clearing nematode infections. Results demonstrated a wide distribution of drug resistance in cattle nematodes. Genetic studies were performed to identify the species of parasites infecting these animals. In nearly all instances, resistance was the result of Cooperia punctata and/or several Haemonchus species. Furthermore, where Cooperia was once considered a minor genus infecting cattle, we have now shown that it has become a predominant pathogen resulting from its resistance to anthelmintics in the face of other nematodes that continue to respond to the presence of the drugs. As a result, overuse of drugs has not only selected for drug-resistant nematodes, but also has selected for species with elevated pathology in the host. Furthermore, this has changed the population dynamics of parasites on pastures. Our results clearly demonstrate that there has been a rapid rise in the prevalence of cattle GI nematodes that are resistant to the most commonly used anthelmintics. This is important and needed information to assist in altering management practices to address the absence of available drug intervention strategies.

2. The ruminant nematodes Ostertagia, Haemonchus, and Teladorsagia down regulate innate immune responses. Methods used by parasites to control host immune responses are poorly understood; however, being able to attenuate these responses is important for establishment of the parasite. The energy molecules ATP and ADP play key roles in the host inflammatory response, and nematodes have been shown to harbor enzymes that can regulate the extracellular levels of these chemicals. To this end, nematode apyrase genes capable of inactivating ATP and ADP were cloned and expressed. We have shown that the parasites secrete these proteins as a way of interfering with the host’s first-line of defense. This occurs during periods of rapid worm growth and tremendous cell damage, but surprisingly, in the absence of inflammation that is an important host mechanism to control infections. As such, it is believed that this is a key method by which the parasite escapes host immunity prior to its emergence as an adult worm, when it causes the greatest damage to the gut. Because this apyrase is secreted and is found within the esophagus of the worm, it provides a viable target for vaccine development.

3. Sequencing of the Ostertagia genome. Efforts were advanced to sequence the Ostertagia genome because it remains the most pathogenic GI nematode of cattle worldwide, causing an estimated $2 billion in lost revenue to producers. The size of the genome is 4X greater than first estimated. Comparisons have been generated between expressed sequences between Ostertagia and Cooperia as a way to better indentify the similarities and differences between these important pathogens of cattle; these comparisons have also included other parasitic nematodes whose genomes and transcriptomes have been sequenced to near completion. Of the genes sequenced, at least 40% are unique to these organisms. These data collectively suggest that sufficient variability exists within this group of organisms to permit identifying and targeting unique genes for future drug intervention strategies or vaccination that will not interfere with host biological functions.

4. Bovine mucin genes and their relationship to protective immune responses have been studied. Mucins are a group of glycoproteins found in secretions that form viscous solutions, and that act as lubricants or protectants on external and internal surfaces of the body. In the bovine GI tract, these mucin genes display a tissue-specific distribution. For example, some membrane-associated mucins are transcribed throughout the whole GI tract; however, in contrast to humans, other mucins are found only in the bovine small and large intestines, but not in esophageal tissue. In response to Ostertagia infection, we found 3 specific mucins, MUC1, MUC6, and MUC20 that were expressed at particularly high levels. Although parasite-induced alterations in mucus biosynthesis start early during infection, the greatest effects were found when adult worms were present on the surface of the abomasal mucosa. We believe that mucin biosynthesis and its regulation plays a critical role in developing first-line, host defense mechanisms against Ostertagia infections. Finding ways to enhance this biosynthesis or methods that the parasite uses to attenuate or circumvent this host activity may provide targets for advancing host protective responses against GI parasites.

Review Publications
Mcclure, M.C., Chung, H.Y. 2010. Characterization of microsatellite loci in the SLA class I region. Genomics. 97:233-234.

Hoorens, P.R., Rinaldi, M., Li, R.W., Goddeeris, B., Claerebout, E., Vercruysse, J., Geldhof, P. 2011. Genome wide analysis of the bovine mucin genes and their gastrointestinal transcription profile. Biomed Central (BMC) Genomics. 12:140.

Li, R.W., Hou, Y., Li, C., Gasbarre, L.C. 2010. Localized complement activation in the development of protective immunity against Ostertagia ostertagi infections in cattle. Veterinary Parasitology. 174(3-4):247-256.

Li, R.W., Li, C., Gasbarre, L.C. 2011. The vitamin D receptor and inducible nitric oxide synthase associated pathways in the development of acquired resistance to Cooperia oncophora infection in cattle. Veterinary Research. 42(1):48.

Rinaldi, M., Dreesen, L., Hoorens, P., Li, R.W., Claerebout, E., Goddeeris, B., Vercruysse, J., Vandenbroeck, W., Geldhof, P. 2011. Infection with the gastrointestinal nematode Ostertagia ostertagi affects mucus biosynthesis in the abomasum of cattle. Veterinary Research. 42(1):61.

Nisbet, A.J., Zarlenga, D.S., Knox, D.P., Meikle, L.I., Wildblood, L.A., Matthews, J.B. 2011. A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses? Parasite Immunology. 33:236-243.

Zarlenga, D.S., Gasbarre, L.C., Nisbet, A.J., Garrett, W.M. 2010. A calcium-activated nucleotidase secreted from Ostertagia ostertagi fourth stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods. Parasitology. 138:333-243.

Last Modified: 4/23/2014
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